Chemical hybridizing agents

ABSTRACT

The present invention relates to a method for inducing male sterility in plants which comprises treating seeds of a plant receptive to said treatment by application of an effective amount of a compound of the formula: ##STR1## wherein R and R 1  are hydrogen, alkyl, alkenl, alkynl, allyl, aryl, halogen or boron and may be the same or different; 
     X and X 1  are fluorine, chlorine, bromine or iodine; nitrate, sulfate, sulfonate, phosphate, citrate, or maleate; 
     n and n 1  are the integers 0 or 1, and may be the same or different; 
     m and m 1  are integers from 0 to 4, and may be the same or different; 
     for a period of time and under conditions sufficient to induce male sterility in adult plants which are grown from said treated seeds.

FIELD OF THE INVENTION

The present invention relates to chemical hybridizing agents; morespecifically it relates to the use of these agents in inducing malesterility in plants.

BACKGROUND OF THE INVENTION

Plant breeders have long been attempting to increase productivity of themore important crops used for food, or for processing as feed, fiber andpharmaceuticals, by their efforts in developing cultivars (cultivatedvarieties) with particularly desirable characteristics. One of the waysin which this aim is frequently accomplished is the development ofsuperior plant lines by infusing desirable traits with already existingcultivars, thus potentially forming a hybrid with exceptionalcharacteristics. The general superiority of F₁ hybrids over either oftheir parents is a widespread phenomenon in a variety of different typesof crops. This superiority may express itself in such features asincreased height, growth rate, leaf area, early flowering and overallhigher yields.

One way in which the production of superior plant lines has beenachieved in the past is by the making of numerous manual crosspollinations to obtain the desired F₁ hybrid. These crosses generallyare carried out between an already existing cultivated crop variety andan unadapted or "wild type" gene donor which possesses one or moretraits which the breeder wishes to incorporate into the cultivatedvariety. Once the production of the F₁ has been accomplished, repeatedbackcrossings and selections are then required to ultimately obtain aplant containing all the characteristics of the cultivated plant as wellas retaining the new, desirable traits introduced from the "wild type"plants. As can easily be seen, this selection procedure is extremelytedious and time consuming; yet, in spite of the difficulties, itremains one of the most widespread of plant breeding techniquescurrently in use.

Because of the problems involved with this method, a number of otheravenues for more efficient production of F₁ hybrids have been and arebeing explored. Among the most avidly pursued fields of endeavor is theconstruction of male sterile lines within the varieties of crop plantsto which hybridization is desired. The principle behind the developmentof male sterile lines is that, in order to produce hybrid seed moreeconomically, the restrictions of controlled cross-fertilization imposedby floral morphology, especially of perfect flowers, must be overcome.To this end, the female parent should be prevented from self- orintraline fertilization. The elimination of self-fertilization requiresandro-self sterility, or the inability of the plant to produce viablepollen. The establishment of the male sterile line thus renders any cropvariety readily adaptable to hybridization with virtually any gene donorhaving the desired characteristics, and eliminates the need forlaborious hand pollination.

Male sterile lines may be established in a number of ways. Handemasculation is one method by which a line may be sterilized. Forexample, large scale production of hybrid corn may be done bydetasselling the female parent; however, the large scale emasculation ofspecies having perfect flowers generally proves to be economicallyunfeasible.

Genetic male sterility is also a known trait, usually inherited as arecessive and monogenic trait in a number of different types of plants.Exploitation of this characteristic is used to produce hybrid seed ofbarley, tomato, pepper, marigold, zinnia, and others. However, there isa basic shortcoming in the use of this technique, in that it isdifficult to obtain a 100% genetic male sterile stand. Overcoming thisdifficulty requires a rather complex use of clever genetic manipulation.Its use, therefore is currently restricted to hybrid seed production ofcultivated plants in which cytoplasmic male sterility has not beenfound, or that in which the male sterile plasmatype exhibits inferioragronomic performance.

Cytoplasmic male sterility provides an additional mechanism forproviding the desired lines for use in hybridization. In this situation,the genetic factors controlling male sterility are found in thecytoplasm. This trait is probably associated with some alteration of thenormal structure or function of mitochondria of plastids. Cytoplasmicmale sterility has found widespread application in the production ofhybrid seed. Widespread production based on this trait is responsiblefor larger percentages of many important cultivated crops such assorghum, sugarbeet, onions, melon, and, most successfully, corn. Anumber of difficulties exist with this system as well. First, it isdifficult to ensure the expression of cytoplasmic male sterility acrossthe range of environments in which hybrid seed may be produced. A femalewheat plant which is 100% sterile in one locality may prove to be only50% sterile in another locality, thus producing obvious difficulties inhybrid seed production.

Furthermore, once the sterile line is established, the female line mustbe maintained through the use of a male fertile maintainer line; andhybrid seed must be restored to at least semi-sterility via a "restorer"line. Clearly the necessary development of effective and appropriatemaintainer and restorer lines presents a considerable obstacle to theefficient and economical exploitation of the trait for the production ofhybrid seed. In fact, a number of important cereal crops, such as wheat,have continued to resist all efforts to establish efficient cytoplasmicmale sterility restorer lines.

A method of producing male sterile lines which circumvents thedifficulties of genetic induction is the use of chemical sterilizationagents. The principle involved here is that the chemical acts as agametocide selectively altering the male gamete, i.e., pollen, byinducing physiological abnormalities, which in turn prevent pollendevelopment, pollen shed, or pollen viability. A number of chemicalcompounds have been shown to have at least a partial effect in producingmale sterility in plants. Among these are: 2-chloroethylphosphonic acid(ethephon; Berhe et al., Crop Science 18: 35-38, 1978); sodium1-(p-chlorophenyl)-1,2-dihydro-4,6-dimethyl-2-oxonicotinate (RH-531+532;Jan et al., Euphytica 23: 78-85, 1974);3-(p-chlorophenyl)-6-methoxy-s-triazine-2,4 (1H,3H)dione-triethanolamine (DPX3778; Johnson et al., Crop Science 18:1026-1028; 2,7-diamino-10-ethyl-6-phenylantridium bromide (ethidiumbromide; Burton et al., Crop Science 16: 731-2, 1976). Although use ofthese compounds obviate the problems encountered with geneticmanipulation, there are still a number of difficulties which might arisewith use of chemisterilants. For example, chemical treatment may resultin induction of only partial sterility, or may be variable in the degreeof male sterility induced under field conditions. They also may producephytotoxic side effects, such as seed shrinking, which may reduce theviability and/or agricultural utility. Further, female sterility mayalso be induced by the use of some of these chemicals. Anotherundesirable feature is that for the most part, these compounds areapplied as foliar sprays. The necessity of such application presents theproblem of environmental pollution, and synchrony with the exact plantstage of development.

It has now been discovered that another class of compounds, DABCO(1,4-Diazabicyclo[2.2.2]octane) and its quaternary salt derivatives havethe unexpected effect of causing male sterility in plants. The subjectcompounds, which are preferably used to treat seeds directly, are thusnot limited to use as foliar chemisterilant sprays, although this is analternate method of application. Use of these compounds for chemicalemasculation allows for the development of all female plants which maybe used to produce large quantities of hybrid seed. These compounds havethe added advantage of producing male sterility in wheat, a plant whichhas traditionally resisted all attempts to establish a successful hybridseed production program.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a method of inducing substantial malesterility in plants which comprises applying to seeds of said plantsreceptive to such induction an effective amount of compounds of theformula: ##STR2## wherein R and R¹ are hydrogen, alkyl, alkenyl,alkynyl, allyl, aryl, halogen or boron and may be the same or different;

X and X¹ are fluorine, chlorine, bromine; iodine; nitrate, sulfate,sulfonate, phosphate, citrate, or maleate;

n and n¹ are the integers 0 or 1, and may be the same or different;

m and m¹ are integers from 0 to 4;

for a period of time and under conditions sufficient to induce malesterility in adult plants which develop from said treated seeds.

Also provided by the present invention is a composition of matter usefulfor inducing male sterility in plants, which comprises an effectiveamount of the above compounds in combination with a carrier solvent.

It is preferred that the alkyl, alkenyl and alkynyl groups have no morethan 6 carbon atoms and that the aryl group be either benzyl or phenyl.

It also provides a method for the production of hybrid seed bycross-breeding male and female parents of different genotype, theimprovement comprising utilizing as the female parent a plant grown fromseed treated with an effective amount of DABCO or one of theDABCO-halogen derivative.

DETAILED DESCRIPTION OF THE INVENTION

The active compounds of the present invention are primarily quaternarysalts of the compound 1,4-diazabicyclo (2.2.2) octane (DABCO). DABCO isa known compound, as are some of its halogen derivatives (see U.S. Pat.No. 3,150,136, the disclosure of which is incorporated herein byreference). Some of the known uses for DABCO compounds are asantibacterial agents, or for ganglionic blocking and as a centralnervous system depressant. It has heretofore not been known to use DABCOor its quaternary salts in the process of inducing male sterility inplants, however. The useful salts of the present invention may bemono-or-di-quaternary salts of DABCO. Particularly preferred are thehalogen derivatives of DABCO, and most preferred are DABCO-benzylchloride, and DABCO-BCl₃. The DABCO compound itself is also contemplatedfor use in the present method.

The present process of male sterility induction is implemented by thetreatment of seeds of a plant possessing characteristics which aredeemed desirable for the specific purpose, with DABCO or one of itsquaternary salts as noted above. The treatment serves, in a manner whichis not yet certain, to prevent pollen formation in the adult plant whichis ultimately derived from a treated seed. Thus is provided a stand ofplants which is functionally all female, and which can then beselectively hybridized by pollination by a plant having differentfeatures which may be desirable to incorporate into the genome of themale sterilized plant. The resulting hybrid seed contains the superiortraits of both the all-female plants and the selected plant by which itwas pollinated. Therefore, the resulting seeds would produce adultplants which are superior to both parent plants in that the desirabletraits of each are combined into a single plant. The present process isparticularly well-suited for plants which are generallyself-fertilizing, such as wheat.

The treatment of the seeds is a relatively simple process. The DABCOsalts are generally dissolved in a hydrocarbon carrier solvent, such ascarbon tetrachloride or benzene, DMSO, or water. Particularly preferredas a solvent is DMSO (dimethylsulfoxide) at a concentration of 0.2-20%preferably about 5%. DMSO is preferred because of its independent effectof inducing some level of male sterility. The active ingredient is addedto the carrier to produce a final concentration within the range of0.01-100 millimolar, with the preferred concentration being about 1 mM.The remaining volume of the solution is made up with double distilledwater. The seeds to be used may be surface sterilized prior to theirtreatment with the chemisterilant. The seeds may be placed in anysuitable container, such as a petri dish, and the active chemicalsolution added. As the present compounds tend to deteriorate with time,and may yield unpredictable or unwanted results when left for longperiods of time, it is generally preferred to use relatively freshpreparations. In some cases, depending upon the thickness of the seedcoat, it is desirable to scarify the seeds to expedite entry of thetreatment solution before germination. The amounts needed for treatmentmay vary, but generally 2 ml/20 seeds is sufficient to produce thedesired result. The seeds may then be covered and incubated in a growthchamber for a period of at least 24 hours. The seeds may then be placedin soil and maintained in a manner consistent with the usual growingconditions for the plant in question. The plants are allowed to mature,and pollination procedures of various types well-known in the field maybe employed upon ripening of the female plant, to produce the desiredhybrid seed.

The treatment prescribed herein may be applied to a wide variety of seedtypes, although, to date, best results have been observed with monocotseeds. The present method has been particularly successful with wheatseeds. However, the process is, in principle, applicable to any type ofplant seed. Whether the present method is useful in producing malesterility in any given seed type may be readily determined by followingthe above-outlined procedure. After the treated seeds have been plantedand grown to maturity, it is a relatively simple matter, particularly inself-fertilizing plants, to observe the seed set of the plants derivedfrom the treated seeds. Absence or reduction of seed set indicates theeffectiveness of inducing sterility by the treatment. Certainmodifications of the abovementioned protocol may be required to obtaincompletely satisfactory results. For example, concentration of theactive ingredient, or length of incubation may have to be varieddepending upon the type of seed used. It is, however, easily within theability of one skilled in the art to determine the appropriateadjustments which will render the treatment most effective.

Many of the compounds to be used in connection with the presentinvention are, as noted above, previously known. They may generally beprepared according by first making the parent molecule, DABCO,anhydrous. This is achieved by sublimation under reduced pressure, inthe presence of a solvent such as dry benzene, dry carbon tetrachlorideor other dry carbon tetrachloride or other hydrocarbons. The appropriatehalide, e.g., boron trichloride or boron trifluoride, is then addeddropwise to the DABCO in an appropriate solvent with stirring, undernitrogen. Complexes generally form within an hour, but reaction timewill depend to some extent on the temperature and the halide used. Thesolid DABCO-halogen complexes are removed by filtration and washed withethyl ether to remove unreacted DABCO and excess carbon tetrachloride,and further washed with distilled water, if necessary, to remove aminesalts. After drying in a vacuum oven, the complexes are stored, in thedark, at 80° F. or less.

Although the mechanism of action of these compounds is not, at thepresent time, determinable with any certainty, it appears probable thatthese chemicals act to alter DNA synthesis, particularly mitochondrialDNA synthesis, in such a manner that the genes which condition malefertility are either rendered non-functional, are preferentiallyunder-replicated, or are preferentially cut out of the DNA and lost.This apparent ability to act as DNA intercalators may indicate apotential utility for the compounds as genetic engineering tools.

The advantages of the use of the present compounds over previously knownchemical sterilizing agents are many. First, they may be applieddirectly to the seeds, rather than as a foliar spray to adult plants.This avoids both the problems of environmental pollution, and thenecessity of synchrony with the exact stage of plant development. Theyhave also proven useful in producing male sterility in wheat, a plantwhich has traditionally resisted efforts to establish male sterilelines. Also, the compounds appear to have little or no effect on femalefertility in plants grown from treated seeds. The present invention willbecome more clearly understood by reference to the followingnon-limiting examples.

EXAMPLE 1

This example illustrates the process of preparing DABCO-borontrichloride.

To a solution of 50 grams of Anhydrous DABCO (Houdry Process andChemical Co., Philadelphia) in 500 ml of carbon tetrachloride, is added117 grams of boron trichloride dropwise with stirring under a drynitrogen atmosphere. At 30° the reaction is complete within about 1.5hours., and the solid boron trichloride may be removed by filtering. Theresulting solid white complex, is then washed with ethyl ether to removeunreacted DABCO and excess carbon tetrachloride, then with water toremove amine salts. The complex is dried at 110° in a vacuum oven, andsubsequently stored in the dark.

Employing analogous procedures, the following compounds have also beenmade.

    ______________________________________                                                 Dabco +            Dabco + 2CH.sub.2 Br.sub.2                                 HF + HCl                                                             Dabco +  Dabco +   Dabco +  Dabco + CH.sub.2 BrCl                             HF       HCl + HI  2Br.sub.2                                                  Dabco +  Dabco +   Dabco +  Dabco + CBr.sub.4                                 2HF      HBr + HI  I.sub.2                                                    Dabco +  Dabco +   Dabco +  Dabco + benzylchloride                            2HCl     CH.sub.3 Cl                                                                             2IBr                                                       Dabco +  Dabco +   Dabco +  Dabco + 1-brom-2-methyl                           2HBr     CH.sub.2 Cl.sub.2                                                                       2BCl.sub.3                                                                             propane                                           Dabco +  Dabco +   Dabco +                                                    2HI      CH.sub.2 Br.sub.2                                                                       allyl                                                                         bromide                                                    Dabco +                                                                       HBR + HF                                                                      ______________________________________                                    

EXAMPLE 2

The following example illustrates the typical experimental procedureused for treating seeds with the compounds of the present invention:

Test chemicals were made to 1.0 millimolar concentration in 5% distilleddimethyl sulfoxide in a total volume of 2 ml. double-distilled water.The control was 5% DMSO solution. Sets of twenty surface-sterilizedwheat seeds (Triticum durum L. cv. `Jori`) were placed in a falconplastic petri dish on a double layer of Whatman No. 2 filter paper. Twoml. of DABCO-BCL₃ was added to each petri dish. The solution beaker wasrinsed with 1.0 ml deonized water, and the washing poured over a thirdfilter paper disc covering the seed. The petri dishes were covered,incubated in a lighted growth chamber for 24 hr., and then placed inplastic pots containing a standard potting soil mix. Each of the groupsof 20 treated seeds were randomly distributed to five pots, four seedsper pot; and the pots were placed randomly on the greenhouse bench.Plants were grown to maturity in a greenhouse, with occasionalsupplemental watering with nutrient solution, and with one chemicaltreatment for insect control. Seeds were allowed to mature, ripen anddry down in the heads prior to harvest.

The results of a typical experiment are outlined in Table 1. Nodifference in time of development, leaf morphology, viability, or plantheight were noted in any treatment, as compared with the control. Testswere also performed to determine the effect of Dabco-boron trichlorideon female fertility. The results presented in Table 1a suggest thatfemale fertility is not substantially affected by the treatment withDabco-halogen complexes.

                  TABLE 1                                                         ______________________________________                                        Effect of DABCO-boron trichloride on reproductive                             properties of greenhouse-grown "Jori" wheat.                                           Number             Number of                                                  of                 sterile  Number of                                         seeds/   Length of spikelet rachis                                   Treatment                                                                              plant    rachis (cm)                                                                             groups/head                                                                            nodes/head                               ______________________________________                                        Control  15.7 a   3.19 a    0.43 a   10.3 a                                   (5% DMSO)                                                                     Dabco-boron                                                                             9.9 b   3.70 a    2.36 b    9.7 a                                   trichloride                                                                   (1 mM)                                                                        ______________________________________                                         Means in columns not followed by the same letter are significantly            different at the 5% level.                                               

                  TABLE1a                                                         ______________________________________                                        Effect of DABCO-boron trichloride on female fertility                         of greenhouse-grown "Jori" wheat.                                                        Number  Weight   Number of                                                   of seed/ of seed/ spikelet Plant                                    Treatment plant    plant (g)                                                                              groups/plant                                                                           Height (cm)                              ______________________________________                                        Control   26.8     1.2      14.8     53.3                                     (DMSO)                                                                        (4 plants)                                                                    DABCO-boron                                                                             34.8     1.4      15.8     56.5                                     trichloride                                                                   (3 mM)                                                                        (4 plants)                                                                    DABCO-boron                                                                             19.0     0.9      13.0     54.2                                     trichloride                                                                   (1 mM)                                                                        (3 plants)                                                                    ______________________________________                                         Pollen from selected untreated plants was used to crossfertilize receptiv     ovules in heads of DABCOcomplex treated plants the day following clipping     of the tops of the lemma and palea to facilitate cross pollination.      

EXAMPLE 3

The preceding protocol was followed in an additional experiment in whichethidium bromide and adriamycin were compared with DABCO-BCl₃ foreffectiveness in reducing seed set. The results are presented in Table2. Although both comparison compounds had a noticeable effect onreduction of seed numbers, only DABCO-BCl₃ showed a statisticallysignificant reduction in seed number.

                  TABLE 2                                                         ______________________________________                                        Effect of chemical seed treatments on seed set and spike                      characteristics of uniculum wheat.                                                                Weight   Number of                                                                              Number                                             Number   of seeds/                                                                              sterile  of rachis                                          of seeds/                                                                              head     spikelet nodes/                                  Treatment  head     (cm)     groups/head                                                                            head                                    ______________________________________                                        Control (5%                                                                              28.8 b   1.44 b   0.95 b   15.9 a                                  DMSO)                                                                         Dabco-BCl.sub.3                                                                          16.3 a   0.96 a   3.50 a   16.4 a                                  Ethidium Bromide                                                                         27.1 b   1.46 b   1.00 b   16.8 a                                  Adriamycin 26.5 b   1.49 b   0.37 b   16.7 a                                  ______________________________________                                         Means in columns not followed by the same letter are significantly            different at the 5% level judged by both Duncan's New Multiple Range Test     and Student NeumannKeuls procedure. All treatments applied in 5% DMSO.   

EXAMPLE 4

The procedure of Example 2 was followed for treatment of barley seeds.The results are summarized in Table 3.

                  TABLE 3                                                         ______________________________________                                        Effect of Dabco-boron trichloride on reproductive                             properties of field-grown "Gus" barley                                                            Number of  Number                                                    Number   sterile    of spikelet                                                                           Plant                                             of seeds/                                                                              spikelet   groups/ Height                                 Treatment  plant    groups/plant                                                                             plant   (cm)                                   ______________________________________                                        Control    32.8     0.5        13.1    31.5                                   (10 plants)                                                                   (deionized H.sub.2 O)                                                         DABCO-boron                                                                              19.8     2.7        11.3    29.3                                   trichloride                                                                   (1 mM) (9 plants)                                                             ______________________________________                                         Seeds were soaked twice as long as those in Table 1.                     

What is claimed is:
 1. A method of inducing male sterility in plantswhich comprises treating seeds of a monocot plant receptive to saidtreatment by application of an effective amount of a compound of theformula: ##STR3## wherein R and R¹ are hydrogen, alkyl, alkenyl,alkynyl, benzyl, phenyl, halogen or boron and may be the same ordifferent and said alkyl, alkenyl and alkynyl have no more than sixcarbon atoms;X and X¹ are fluorine, chlorine, bromine or iodine;nitrate, sulfate, sulfonate, phosphate, citrate, or maleate; n and n¹are the integers 0 or 1, and may be the same or different; m and m¹ areintegers from 0 to 4, and may be the same or different; for a period oftime and under conditions sufficient to induce male sterility in adultplants which develop from said treated seeds.
 2. The method of claim 1wherein the plant is wheat.
 3. The method of claim 1 wherein thecompound is DABCO-BCl₃.
 4. The method of claim 1 wherein the compound isDABCO-benzyl chloride.
 5. A selective male sterilant composition usefulin induction of male sterility in plants which comprises an effectiveamount of at least one compound of the formula: ##STR4## in combinationwith DMSO as a carrier solvent, wherein R and R¹ are hydrogen, alkyl,alkenyl, alkynyl, benzyl, phenyl, halogen or boron and may be the sameor different and said alkyl, alkenyl and alkynyl have no more than sixcarbon atoms;X and X¹ are fluorine, chlorine, bromine or iodine;nitrate, sulfate, sulfonate, phosphate, citrate, or maleate; n and n¹are the integers 0 or 1, and may be the same of different; m and m¹ areintegers from 0 to 4 and may be the same of different.
 6. A method ofinducing male sterility in plants which comprises treating a plantphysiologically committed to flowering by foliar application of aneffective amount of a compound of the formula: ##STR5## wherein R and R¹are hydrogen, alkyl, alkenyl, alkynyl, benzyl, phenyl, halogen or boronand may be the same or different and said alkyl, alkenyl and alkynylhave no more than six carbon atoms;X and X¹ are fluorine, chlorine,bromine or iodine; nitrate, sulfate, sulfonate, phosphate, citrate, ormaleate; n and n¹ are the integers 0 or 1, and may be the same ordifferent; m and m¹ are integers from 0 to 4, and may be the same ordifferent; for a period of time and under conditions sufficient toinduce male sterility in adult plants which develop from said treatedplants.